Chemistry And The Aquarium: Iron: A Look At Organisms Other Than Macroalgae

I do believe, however, that the benefits outweigh the risks when one is growing macroalgae. To date there have been no claims of a demonstrated problem in a reef tank from reasonable iron additions.

Contents

In my article two months ago, I discussed iron in reef tanks. Much of that article was directed toward the effect that iron has on macroalgae, and in that article I concluded that it was generally positive. Of course, macroalgae are not the only things in our tanks, and if iron is having an effect on other organisms, it is important to understand it. Some hobbyists, for example, have suggested that iron may “supercharge” cyanobacteria, while others have suggested just the opposite. It has also been suggested that iron supplements may actually be “toxic” to corals when dosed to a reef tank. In looking at the literature, it would appear that diatoms and other microorganisms can also be impacted by iron additions.

Unfortunately, definitive research that can be extrapolated to aquaria is lacking. Most studies of iron as it relates to growth and toxicity are directed toward organisms in natural settings like the ocean, and not reef tanks. Unfortunately for reefkeepers, the conditions in reef tanks may be sufficiently different than in the ocean as to make extrapolations of these studies to reef tanks ambiguous. In a situation such as this, I think it best if hobbyists understand the scientific basis for these various concerns, and can then use the information to make up their own minds as to its importance.

In this article, I will review some of the pertinent scientific literature in this area, including one paper that has been suggested to show iron “toxicity” to a coral that many reefkeepers maintain. Based on the papers described here, I’ll also suggest some things to look for if you are dosing iron and are worried about problems that it might cause.

Speciation of Iron in Seawater

Before proceeding to discuss the effect of iron on various marine organisms, there are some important issues that relate to the bioavailability of iron that should be understood. Bioavailability is a concept that has applications that range from medicine to ecology. In short, its premise is that not all of some particular agent is available to organisms. For example, if a pharmaceutical that is intended to treat brain cancer is given orally and is not absorbed from the gastrointestinal tract, it will not likely be effective. In this case, the drug would be described as having poor bioavailability. That is, it is not available where it is needed.

In discussing metals such as iron in seawater, bioavailability can be critically important. Iron can take many forms in seawater, some of which are readily available to organisms and some of which are not. For many organisms, iron that is complexed to certain organic ligands is not bioavailable. In a sense, the organisms cannot use the iron, and are not impacted by it. In other cases, the organic/iron complex can be absorbed and used, and in some of the most interesting cases, these ligands are specifically designed by organisms to “go out and collect iron”. Consequently, it behooves us to understand as much as possible about the speciation of iron in seawater (and tank water) in order to understand the effects that it might be having on organisms.

While these general concepts apply to all metals in seawater, it has recently become clear that iron in particular is extensively bound to organic chelators. For example, one research group recently claimed in the journal Nature that “Dissolved Fe(III) in the upper oceans occurs almost entirely in the form of complexes with strong org. ligands presumed to be of biol. origin.” 1

The chelators that bind iron in seawater (and by analogy, reef tank water) are many, and come from many sources that are present in our reef tanks. One researcher, for example, details the concern: “The present report shows that both inorganic Fe(III) in the presence of oxygen, and humic Fe(III) which stimulates lipid peroxydation, trigger or stimulate the release of chelators from green algae, red algae, and cyanobacteria.” 2 Consequently, we should anticipate that we have such chelators in our tanks.

Moreover, some researchers claim that the speciation of iron in seawater is more important than the total concentration. That is, one cannot just determine to total iron concentration to know whether it is of adequate bioavailability for any given organism. One must know what organic ligands are bound to it: “Recent observations showed that, more than its absolute quantities in surface waters, the biological availability of iron through chelation with complexing ligands could in fact limit the biomass growth. Thus, the study of organic speciation of iron in sea-water becomes a necessary step, if we aim to better understand the link between the climate forcing and iron dynamics in the oceans”. 3

Others researchers claim “Previous coastal marine studies concluded that total dissolved Fe may affect initiation of algal blooms, including brown tides of Aureococcus anophagefferens. However, the existence of unavailable colloidal and organically-complexed Fe make the dissolved pool a poor indicator of what is bioavailable for phytoplankton assimilation.”4

Consequently, if one were assessing the status of a tank with respect to iron, one would have to understand the specific nature of the iron present. In other words, one cannot use measurements of total iron, such as those provided by most test kits or by the analytical technique ICP, to know whether a given tank “needs” iron or not. This unfortunate circumstance prohibits reefkeepers dosing iron from having the type of simple relationship between concentration and effect that is enjoyed by many other ions of interest, such as calcium. One might be able to use these techniques to follow bioavailability as a function of time in a single reef tank with increases or decreases in iron dosing since the level of available chelators may remain mostly unchanged (but I have not done this experiment).

Whole System Studies in the Ocean

The iron enrichment literature as a whole falls into 3 categories. In the first, individual species are studied in isolation. In the case of microorganisms, these studies would be the least relevant to aquaria where there is competition between many organisms for the same nutrients, space, etc, as well as predation. The second category examines the effect of iron in the open ocean, but does not compare changes in growth of one organism to changes in growth of other organisms. In the third category are those studies that have added iron to systems (such as the ocean) and looked at the absolute AND the relative populations of various organisms, including the effects of predation.

It is this last category of study that I will describe first, since it most accurately reflects what one might expect to happen when one adds iron to a whole ecosystem: a reef tank. Unfortunately, there are no studies of this kind that describe the effects of iron on corals. Consequently, the effects of iron on corals will have to be inferred from less grand studies.

In one study, the researchers added iron to a variety of ocean environments and tracked the population change in various organisms.5 What they found is very interesting. The primary organisms that increased relative to the others were diatoms. The also found that cyanobacteria and dinoflagellates declined, and bacteria remained largely unchanged. They state “…at least eight diatom genera and an undetermined number of different autotrophic nanoplankton taxa were present in higher numbers in the Fe+ carboys, whereas cyanobacteria, one diatom group, and dinoflagellates were more numerous in the controls”. Also, “In contrast [to studies adding things besides iron], the HNLC bacterial communities in our experiments showed only a small response, despite large Fe-induced changes in biological and biochemical parameters.”

Similar results were seen in the well-publicized IRONEX experiments. These studies have shownthat when there is adequate nitrate and phosphate, iron may become the limiting factor for phytoplankton growth. 6-10 In IRONEX II, for example, diatoms accounted for 17% of the biomass growth prior to iron enrichment, and 79% after iron addition.11 In this test, in fact, diatoms experienced a 70x increase in population.12 It was also found in this study that cyanobacteria did not increase growth as much as diatoms. One common cyanobacterium, Prochlorococcus, actually decreased.13 Heterotrophic (nonphotosynthetic) dinoflagellate and ciliate populations increased substantially (>3x), presumably as they consumed the diatom bloom. 12 Heterotrophic bacteria experienced a smaller population increase (1.7 x).14

On the basis of these types of studies, the literature points to diatoms as one of the most likely things to become relatively enhanced by iron additions.

Cyanobacteria and Iron

It has been suggested by some hobbyists that iron additions to reef tanks may drive the growth of cyanobacteria, and that one should not dose it for that reason. I’ve not seen that effect in my tank, nor have I heard it reported in others. It was also not reported to be significant in the open ocean studies described above. Nevertheless, since cyanobacteria are a problem for many reefkeepers, this issue is an especially important one to address.

Fortunately, there is a great deal of literature on the relationship between iron and cyanobacteria. Most of the literature indicates that cyanobacteria are especially well suited to low iron environments because they are able to release siderophores that bind to iron and give them a competitive advantage over other organisms. This is, in fact, one of the reasons why they have been studied so extensively with respect to iron. If they do generally have a competitive edge at low iron levels, then adding iron supplements and swamping out this competitive advantage may make strategic sense if reducing cyanobacteria is a goal.

In one paper, for example, the researchers conclude “that cyanobacteria are efficiently adapted to grow in low-Fe environments (providing sufficient light for photosynthesis is available)…” 15 In a different paper, the researchers state: “This review focuses on how cyanobacteria respond to growth-limiting levels of available iron and on how siderophores potentially alter the biological availability of iron in the system thereby allowing the cyanobacteria to exist at low iron availabilities.” 16 In another paper the researchers show that “The growth rates and intracellular and total cellular

iron levels for Synechococcus PCC 7002 demonstrate that iron availability does not directly dictate the maximum growth rate of these cyanobacteria…”17

Finally, in this paper, it appears that the three species of cyanobacteria tested are not iron limited for growth in the ocean (2 are phosphate limited, the other may be phosphate limited) while one of the species of macroalgae may be iron limited (Dictyota bartayresiana). 18

To be fair, some researchers do make seemingly contrary claims, though the fact that there are many species of cyanobacteria makes that result not overly surprising. One research group states that “Our results suggest that in 75% of the global ocean, iron availability limits nitrogen fixation by this organism.”19 Previously, other researchers had shown that iron does indeed stimulate growth of this particular cyanobacterium.20

These results for cyanobacteria are essentially what one would expect for an organism that can grow well even in low iron conditions. This fact does not demonstrate that the cyanobacteria won’t become iron limited under the higher phosphate and nitrate conditions present in a reef tank, but it also does not indicate that there is, at present, any cause for alarm about cyanobacteria and iron additions.

Effects of Iron on the coral Stylophora pistillata

Unlike the research areas described above, the work on corals has been very limited, with three papers meriting attention. The first and most important of these papers, Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment, was published in the Journal of Experimental Marine Biology and Ecology.21 The experiments in this paper essentially consisted of putting coral fragments (7 cm long) in a series of tanks. The fragments were monitored for 4 weeks, the water was altered chemically, and the fragments were monitored for another 3 weeks. This monitoring consisted of several measurements: coral growth rate, zooxanthellae density, photosynthesis rate, and chlorophyll content.

The starting water itself was pumped from the Mediterranean. It is low in nutrients, such as nitrate (< 0.06 ppm; < 1 mM); phosphate (< 0.02 ppm< 0.2 mM), and iron (< 0.2 ppb; < 4 nM). For test purposes, iron was raised to 0.3 ppb, and nitrate was raised to 0.15 ppm (yes, even the enriched nitrate level was far below that in most reef tanks). The water volume of each tank was replaced continually so that a 100% change took place each hour.

Let’s begin, as they did, by discussing the zooxanthellae density. This density is a measure of the number of zooxanthellae cells present. It can be calculated as the number of zooxanthellae per animal cell, or by the total density of zooxanthellae. By both of these measures, the zooxanthellae increased substantially in all of the test aquaria compared to the control. The largest increase in zooxanthellae was found in that with just iron additions. Essentially the same results were found for the chlorophyll content. On the face of it, these experiments suggest that the growth rate of the zooxanthellae is both iron and nitrate limited.

I’ll discuss later whether it is advantageous or not to boost zooxanthellae growth rates, but from this experiment is not clear is whether the iron and nitrate additions cause ongoing growth of the zooxanthellae, with excess zooxanthellae continually being expelled or otherwise lost, or whether the zooxanthellae have simply multiplied and reached a new plateau in density where they continue to grow at rates similar to before the iron and nitrate enrichment. This issue is important because it bears on coral growth rates (below) and whether the changes seen in this 3 week test will continue for additional time periods.

The authors also measured various aspects of photosynthesis for all of these corals. They found that photosynthesis was increased in all of the test tanks, with iron alone being the largest increase. On a per zooxanthellae basis, however, the photosynthesis was unchanged. This suggests that more zooxanthellae photosynthesize more as a whole (just as a large field of corn photosynthesizes more than a small field), but that the individual cells are photosynthesizing at about the same rate. Apparently the added nutrients have increased the numbers of zooxanthellae, but did not otherwise impact photosynthetic activity.

Finally, the researchers measured coral growth by weight. The growth rate was found to decrease substantially in each of the test tanks compared to control. That is, iron and nitrate, individually and in combination, had a big effect on coral growth rates, with all of them reducing growth by about 30%. The authors state that “Iron seems, therefore, toxic to the coral host, even if it increases the total number of algae.”

The nature of this “toxicity”, however, may be somewhat less worrisome to reefkeepers than the word implies. They authors propose a number of mechanisms for this effect, of which they seem to prefer the simple suggestion that if the zooxanthellae are growing more rapidly, they won’t be delivering as much in the way of useful photosynthetic byproducts to the host animal, decreasing its growth rate. The do state that the relationship between calcification and iron additions remains unclear.

This explanation begs the question, suggested above, of whether this was a one time change caused by a one time increase in zooxanthellae, or whether there will be an ongoing lower level of delivery of photosynthetic byproducts. Only longer-term experiments would easily answer this question.

I have no reason to doubt the possibility of reduced delivery of photosynthetic byproducts, and it makes perfect sense, but I’d like to propose another possible explanation that they did not address: phosphate limitation. For phytoplankton, phosphorus limitation in the ocean is usually secondary to nitrogen limitation. That is, nitrogen is more limiting. Still, according to Millero22:

“Above a phosphate concentration of 0.3 mM the rate of growth of many species of phytoplankton is independent of the concentration of P. Below 0.3 mM cell division becomes inhibited and P-deficient cells are produced. This probably does not occur in the oceans since NO3- is usually exhausted before PO4—- falls to this critical level”

The same is largely true of most macroalgae, with nitrate and phosphate being the limiting factors under different conditions and for different species.23-25 Of course, those studies relate to phytoplankton and macroalgae, and not a coral. However, it is not an unreasonable hypothesis that corals may be similarly limited at the low levels of phosphate encountered in this test (< 0.2 mM) when given extra nitrate.

Additionally, the IRONEX studies have shown that when there is adequate nitrate and phosphate, iron may become the limiting factor for phytoplankton growth.6-10 In IRONEX I, for example, the phosphate level was1 mM and nitrate was 12 mM, both well above the values in this test, even after nitrate enrichment. In that experiment, iron appeared to limit phytoplankton growth.

Putting these two ideas together, and retaining the caveat that these various studies involved phytoplankton and macroalgae, and not a coral, it is a reasonable possibility that the corals in this study with enriched iron and nitrate may be phosphate limited. In such a scenario, the rapidly growth zooxanthellae may use up the absorbed phosphate, making the host unable to obtain enough phosphate, and thereby grow more slowly.

If phosphate limitation is a factor here, then it may not be so in reef tanks, where phosphate levels are typically far higher than in this test (and often equivalent to that in the IRONEX I study). Additionally, since nitrate alone at 0.15 ppm had almost as big of an effect as nitrate plus iron, then perhaps all, or nearly all reef tanks (those with nitrate above 0.15 ppm) are already experiencing this reduced coral growth rate due to increased zooxanthellae growth, and the iron may not make the problem substantially worse. Moreover, there may already be more than enough iron in reef tanks for this effect to have happened even in the absence of any iron supplements.

Whether one is concerned with coral growth rates, or even zooxanthellae density (which may impact the colors of corals), there is obviously substantial uncertainty here with any extrapolation of these results to real reef tanks. Nevertheless, the experiment is interesting and helps us to understand issues involving iron in our reef tanks better. In particular, it helps guide us in what types of problems to be on the lookout for. These potential effects are detailed at the end of this article.

Effects of Iron on Other Corals

A similar study of iron addition is described in a second paper titled Metal tolerance in the scleractinian coral Porites lutea.26** **Surprisingly, it seems to suggest quite the opposite of the paper described above. They claim “Exposure of the scleractinian coral P. lutea to elevated iron concns. leads to a loss of zooxanthellae (symbiotic algae) from the coral tissues”. They go on to state that corals seem to adjust to elevated iron levels, so that the effect on the zooxanthellae is reduced over time. It is not clear whether the different conclusion obtained here compared to that in the Stylophora pistillata paper is due to differences between the species of coral tested, or some other aspect of the study methodology.

A third paper on corals is titled Indications from photosynthetic components that iron is a limiting nutrient in primary producers on coral reefs. 27 * *This paper discusses biochemical evidence of iron limitation in a variety of organisms on a coral reef, but does not actually test elevated iron levels to see if growth limitation is real. In it, these authors suggest that zooxanthellae in Sinularia sp. may be iron deficient. They conclude “The degree and extent of Fe-stress in primary producers on a coral reef thus may influence growth rates, biomass, and distribution of species”.

In addition to these papers on coral growth, there are many papers on the presence of iron in coral tissue28 and skeletons.29, 30 These papers frequently show that iron levels in corals are increased when the level of iron in the water is increased. Whether that is generally “good or bad” is not typically addressed.

Things to watch for if dosing iron

The research described in this article has suggested a variety of things to watch for if dosing iron. I’ve not noticed any of them in my tank, but I’m not sure that given my particular tank, I would have detected some of them anyway. If anyone does initiate iron dosing in their tanks and notices one of these, I’d appreciate hearing of it.* *

If the increase in zooxanthellae described above happened in some of the brightly colored corals that we keep, it is possible that the color may be dimmed toward brown.

If the decrease in zooxanthellae described above happened in some of the Sinularia sp. that we keep, it is possible that the color may be lightened or brightened.

If the increase in zooxanthellae described above happened in an ongoing fashion, it is possible that aquarists may observe the expulsion of zooxanthellae more frequently.

If the reduced coral growth rates described above translated to reef tanks, then it is possible that certain corals may grow substantially more slowly when dosing iron than when not. Whether this is good or bad may depend on whether the aquarist likes to collect coral fragments from the tank or not.

If, in fact, iron has any effect on cyanobacteria in a reef tank, then aquarists may experience decreased (or increased) levels of it.

Diatoms may begin to grow more rapidly if they are iron-limited in reef tanks.

Photosynthetic dinoflagellates may decline in population, presumably as other organisms better compete against them, though heterotrophic dinoflagellates may increase in response to food (diatom) availability.

Given these various concerns, and the fact that we do not really know how these experiments translate to a reef tank, I would, at present, not recommend significant iron additions for reef tanks with no macroalgae. I made this point in the previous article on the basis of driving microalgae growth when there is no macroalgae to take up the nutrients. The topics discussed in this article serve to support the contention that there is little reason to add iron in the absence of macroalgae.

I do believe, however, that the benefits outweigh the risks when one is growing macroalgae. To date there have been no claims of a demonstrated problem in a reef tank from reasonable iron additions. Now that people have some clues as to what to look for, however, we may have such reports in the future.

References

Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment. Ferrier-Pages, Christine; Schoelzke, Vanessa; Jaubert, Jean; Muscatine, Len; Hoegh-Guldberg, Ove. Observatoire Oceanologique Europeen, Centre Scientifique de Monaco, Monaco, Monaco. Journal of Experimental Marine Biology and Ecology (2001), 259(2), 249-261. For those without a subscription or a handy library that has it, it is available online for $30 for most individuals (some people seem to get a different message at that site and I don’t understand why; my two computers even get different messages; this may relate to whether the computer is “thought” by the web site to be part of an institution that does or does not already have a relationship with the publisher).